Mitochondria-dependent apoptosis is a major cell death pathway necessary for ensuring normal development and maintaining tissue homeostasis. Abnormal blockade of this pathway is required for tumor formation and cancer metastasis. On the other hand, the core machinery in this pathway is required for the actions of many cancer therapeutics. The Bcl-2 family proteins, including the anti-apoptotic members, the effectors (Bax and Bak), and the pro-apoptotic BH3-only proteins, are the major regulators and effectors of mitochondrial outer membrane permeabilization (MOMP), a central control point leading to apoptosis. Through biochemical and genetic studies, it has been established that Bax and Bak are two essential effectors of MOMP, and are activated during apoptosis. The mechanism of Bax/Bak activation has been considered the life-to-death switch of the cell, and has been intensively investigated in the past two decades. Recently, an overwhelming consensus has been reached to support the Direct Activation model, in which Bax/Bak activation is primarily triggered by three ?direct activator? BH3-only proteins, Bid, Bim, and Puma, which transiently engage and attack Bax/Bak during apoptosis. However, in our preliminary studies, we found that none of these three proteins are necessary for the activation of Bax/Bak and apoptosis once the anti-apoptotic Bcl-2 proteins are suppressed. As these results are incompatible with the Direct Activation model, we propose to examine the mechanism of Bax/Bak activation in the absence of Bid, Bim, and Puma. We hypothesize that the direct activation activities of all putative direct activator BH3-only proteins are not necessary for Bax/Bak activation. Following a BH3-only protein-mediated neutralization of the anti-apoptotic Bcl-2 proteins, Bax/Bak activation occurs spontaneously. In addition, Bid, Bim, Puma or p53 may have a modulatory function in accelerating Bax/Bak activation following neutralization of the anti-apoptotic Bcl-2 proteins Using genetics, cell biology, and biochemical interaction studies, the following three specific Aims are proposed to investigate the mechanism of Bax/Bak activation without Bid, Bim, and Puma. In Aim 1, we will investigate the role of Bid, Bim, Puma, and p53 in the kinetics of Bax/Bak activation after neutralization of the anti-apoptotic Bcl-2 family proteins. In Aim 2, we will examine the involvement Bik, Bmf, Noxa, and Hrk in Bid/Bim/Puma-independent Bax/Bak activation. Overall, we propose to investigate the mechanism of Bax/Bak activation in the absence of Bid, Bim, and Puma following neutralization of anti-apoptotic Bcl-2 family proteins in cancer cells. Results from the proposed studies should either significantly modify the current model, or provide critical clues to novel and physiologically relevant models of Bax/Bak activation.